The invention relates to proteins involved in neurotrophin signaling.
The regulation of cell growth and survival is believed to be under the control of a wide variety of signaling cascades. One such cascade is that which transduces the presence of a neurotrophin ligand. Early in vivo and in vitro experiments demonstrated that nerve growth factor (NGF) plays critical roles in the development of the nervous system. The cloning of brain-derived neurotrophic factor (BDNF) subsequently revealed a homology with NGF that spurred the cloning and characterization of neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5) and neurotrophin-6 (NT-6). Each of these proteins promotes survival of specific populations of neurons and affects aspects of the neuronal phenotype.
The mammalian neurotrophins interact with two types of cell surface receptors. The trk receptors (e.g., trkA, trkB, and trkC) are highly-related transmembrane receptor tyrosine kinases, each of which preferentially binds one or a subset of the neurotrophin family members trk receptors play a critical role in mediating the effects of the neurotrophins, and their activation results in the effects typically associated with neurotrophin action.
The p75NTR receptor (p75NTR), which binds all neurotrophins with approximately equal affinity, is a member of the tumor necrosis factor (TNF) receptor superfamily. In contrast to the rapid progress made in elucidating the mechanism of action of the trk receptors, the physiological roles of the p75NTR have been more difficult to discern. At present, the actions of p75NTR fall into two categories. First, p75NTR appears to functionally collaborate with trk receptors to either enhance or reduce neurotrophin-mediated trk receptor activation. Second, p75NTR acts autonomously to activate signaling cascades that may be involved in apoptosis and inflammation.
A number of proteins which directly interact with the intracellular domains of members of the TNF receptor superfamily have been identified, including TRADD, FADD, and members of the TRAF family. Generally, the intracellular domain of p75NTR is highly conserved across species, but not conserved with other members of the TNF receptor superfamily. The exception to the general lack of homology between p75NTR and the TNF receptor superfamily is the presence of a 90 amino acid stretch termed the xe2x80x9cdeath domain.xe2x80x9d In TNF receptor-1 (TNFR-1), fas, DR3, and other related receptors, the death domain is required to mediate interactions with either FADD or TRADD. We have previously found, however, that the p75NTR does not bind TRADD or FADD proteins. The failure of p75NTR to bind FADD or TRADD is likely due to the fact that the tertiary structure of the p75NTR death domain differs considerably from these other, receptors, suggesting that p75NTR must facilitate apoptosis through another mechanism. Thus, the mechanism by which neurotrophins signal via p75NTR to modulate apoptosis remains unknown.
There is a need to identify additional components of the neurotrophin signal transduction pathway. These components would be useful as targets for pharmacologic intervention in patients diagnosed with an apoptotic disease.
In a first aspect, the invention features a substantially pure NRAGE polypeptide (previously referred to as PRI-MAGE). In a preferred embodiment, the polypeptide is from a mammal (e.g., a human). In a second preferred embodiment, the polypeptide binds to p75NTR.
In a second aspect, the invention features a substantially pure polypeptide having 50% or greater amino acid sequence identity to the amino acid sequence of the protein of the first aspect and binds to p75NTR. Preferably, the polypeptide has 70% or greater amino acid sequence identity to the amino acid sequence of the protein of the first aspect and binds to p75NTR. More preferably, the polypeptide has 85% or greater amino acid sequence identity to the amino acid sequence of the protein of the first aspect and binds to p75NTR.
In a third aspect, the invention features a substantially pure polypeptide that modulates apoptosis, wherein the polypeptide has 50% or greater amino acid sequence identity to an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
Preferably, the polypeptide has 70% or greater amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. More preferably, the polypeptide has 80% or greater amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
In a fourth aspect, the invention features a substantially pure nucleic acid molecule encoding an NRAGE polypeptide. In one embodiment, the nucleic acid molecule is from a mammal (e.g., a human). In another embodiment, the nucleic acid is cDNA.
In a fifth aspect, the invention features substantially pure DNA having a sequence of FIG. 1C (SEQ ID NO: 3) or FIG. 1D (SEQ ID NO: 4), or degenerate variants thereof, and encoding an amino, acid sequence of FIG. 1A (SEQ ID NO: 1) or FIG. 1B (SEQ ID NO: 2).
In a sixth aspect, the invention features substantially pure DNA that binds at high stringency to a DNA sequence of FIG. 1C (SEQ ID NO: 3) or FIG. 1D (SEQ ID NO: 4).
In a seventh aspect, the invention features purified DNA sequence substantially identical to a DNA sequence shown in FIG. 1C (SEQ ID NO: 3) or FIG. 1D (SEQ ID NO: 4).
In one embodiment, the nucleic acid is operably linked to regulatory sequences for expression of the polypeptide encoded by the nucleic acid and wherein the regulatory sequences comprise a promoter. Preferably, the promoter is a constitutive promoter, is inducible by one or more external agents, or is cell-type specific.
In an eighth aspect, the invention features a vector that includes the nucleic acid of the seventh aspect, the vector being capable of directing expression of the polypeptide encoded by the nucleic acid in a vector-containing cell.
In a ninth aspect, the invention features a cell expressing the nucleic acid of the seventh aspect.
In a tenth aspect, the invention features a method for identifying a compound that modulates binding of NRAGE to p75NTR. The method includes: (a) providing a cell expressing an NRAGE polypeptide; (b) contacting the cell with a candidate compound; and (c) monitoring the level of binding of the NRAGE polypeptide to p75NTR, wherein a change in the level of the binding in response to the candidate compound relative to a level of binding in a cell not contacted with the candidate compound indicating the presence of a compound that modulates binding of NRAGE to p75NTR. Preferably, the cell is from a mammal (e.g., a human or a rodent).
In an eleventh aspect, the invention features a kit for determining the amount of NRAGE polypeptide in a sample, the kit including a substantially pure antibody that specifically binds an NRAGE polypeptide. Preferably, the kit further includes a means for detecting the binding of the antibody to the NRAGE polypeptide.
In a twelfth aspect, the invention feature a substantially pure antibody that specifically binds to an NRAGE polypeptide. In one embodiment, the antibody is selected from a group consisting of a polyclonal antibody, a monoclonal antibody, and a neutralizing antibody.
In a thirteenth aspect, the invention features a method for modulating cell apoptosis, the method includes administering an NRAGE polypeptide to the cell.
In a fourteenth aspect, the invention features a method for modulating apoptosis. The method includes administering to the cell a compound that modulates binding of NRAGE to p75NTR. In various embodiments, the compound is a chemical, a drug, or an antibody that specifically binds to an NRAGE polypeptide. A preferred antibody is a neutralizing antibody. In one preferred embodiment, the compound is an NRAGE antisense nucleic acid molecule.
In preferred embodiments of the thirteenth or fourteenth aspect, the cell is in a mammal (e.g., a human or a rodent).
In another embodiment of the thirteenth or fourteenth aspects, apoptosis is decreased in a mammal diagnosed as being HIV-positive, or as having AIDS, cirrhosis of the liver, a neurodegenerative disease, a myelodysplastic syndrome, or an ischemic injury.
In yet another embodiment of the thirteenth or fourteenth aspects, apoptosis is increased in a mammal diagnosed as having a cell proliferation disease.
In a fifteenth aspect, the invention features a therapeutic composition having as an active ingredient an NRAGE polypeptide, an antibody that specifically binds an NRAGE polypeptide, or an NRAGE antisense nucleic acid molecule, the active ingredient being formulated in a physiologically acceptable carrier. Preferably, the composition modulates cell apoptosis.
In a sixteenth aspect, the invention features a method for identifying a compound that modulates p75NTR biological activity. The method includes (a) providing a cell expressing an NRAGE polypeptide; (b) exposing the cell to a candidate compound; and (c) monitoring the level of expression of the NRAGE polypeptide, a change in the level of expression in response to the candidate compound relative to a level of expression in a cell not contacted with the candidate compound indicating the presence of a compound that modulates p75NTR biological activity.
In a seventeenth aspect, the invention features a method for identifying a compound that modulates p75NTR biological activity. The method includes (a) providing a cell expressing an NRAGE polypeptide capable of binding to p75NTR; (b) contacting the cell with a candidate compound; and (c) monitoring the level of the binding of the NRAGE polypeptide to p75NTR, a change in the level of binding in response to the candidate compound relative to a level of binding in a cell not contacted with the candidate compound indicating the presence of a compound that modulates p75NTR biological activity.
In a preferred embodiment of the sixteenth or seventeenth aspect, the cell is from a mammal (e.g., a human or a rodent).
By xe2x80x9cpolypeptidexe2x80x9d is meant any chain of more than two amino acids, regardless of post-translational modification, such as glycosylation or phosphorylation.
NRAGE polypeptides that are a part of the invention include those polypeptides that bind to p75NTR, are capable of binding an antibody which specifically binds NRAGE, or modulate p75NTR biological activity in a cell. Preferred NRAGE polypeptides are those represented by the amino acid sequences of SEQ ID NO: 1 (FIG. 1A) and SEQ ID NO: 2 (FIG. 1B).
NRAGE nucleic acids that are a part of the invention include those nucleic acids encoding polypeptides that bind to p75NTR, are capable of binding an antibody which specifically binds NRAGE, or modulate p75NTR biological activity in a cell. Preferred NRAGE nucleic acids are those represented by the nucleotide sequences of SEQ ID NO: 3 (FIG. 1C) and SEQ ID NO: 4 (FIG. 1D).
By xe2x80x9csubstantially identicalxe2x80x9d is meant a polypeptide or nucleic acid exhibiting at least 50%, preferably 85%, more preferably 90%, and most preferably 95% identity to a reference amino acid or nucleic acid sequence. For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids. For nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides.
Sequence identity is typically measured using sequence analysis software with the default parameters specified therein (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). This software program matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
By xe2x80x9chigh stringency conditionsxe2x80x9d is meant hybridization in 2xc3x97SSC at 40xc2x0 C. with a DNA probe length of at least 40 nucleotides. For other definitions of high stringency conditions, see F. Ausubel et al., Current Protocols in Molecular Biology, pp. 6.3.1-6.3.6, John Wiley and Sons, New York, N.Y., 1994, hereby incorporated by reference.
By xe2x80x9cp75NTR biological activityxe2x80x9d is meant binding to NRAGE, modulating p75NTR-mediated apoptosis, or any other p75NTR-mediated activity known in the art. Additional examples are provided, for example, in Barker (Cell Death Differ. 5:346-356, 1998) and Miller and Kaplan (Cell Death Differ. 5:343-345, 1998).
By xe2x80x9csubstantially pure polypeptidexe2x80x9d is meant a polypeptide that has been separated from the components that naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the polypeptide is an NRAGE polypeptide that is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, pure. A substantially pure NRAGE polypeptide may be obtained, for example, by extraction from a natural source (e.g., a neuronal cell), by expression of a recombinant nucleic acid encoding an NRAGE polypeptide, or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
A polypeptide is substantially free of naturally associated components when it is separated from those contaminants that accompany it in its natural state. Thus, a polypeptide which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components. Accordingly, substantially pure polypeptides include those which naturally occur in eukaryotic organisms but are synthesized in E. coli or other prokaryotes.
By xe2x80x9csubstantially pure nucleic acidxe2x80x9d is meant nucleic acid that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid of the invention is derived, flank the nucleic acid. The term therefore includes, for example, a recombinant nucleic acid that is incorporated into a vector; into an autonomously replicating plasmid or virus; into the genomic nucleic acid of a prokaryote or a eukaryote cell; or that exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant nucleic acid that is part of a hybrid gene encoding additional polypeptide sequence.
By xe2x80x9cantisense,xe2x80x9d as used herein in reference to nucleic acids, is meant a nucleic acid sequence that is complementary to the coding strand of a gene, preferably, an NRAGE gene. Preferably the antisense nucleic acid molecule decreases the amount of transcription from the gene; more preferably, the decrease is at least 10%, and most preferably, the decrease is at least 50% when administered at the maximally effective dose.
By xe2x80x9csubstantially pure antibodyxe2x80x9d is meant antibody which is at least 60%, by weight, free from proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably 90%, and most preferably at least 99%, by weight, antibody. A purified antibody may be obtained, for example, by affinity chromatography using recombinantly-produced protein or conserved motif peptides and standard techniques.
By xe2x80x9cspecifically bindsxe2x80x9d is meant an antibody that recognizes and binds to, for example, a human, mouse, or rat NRAGE polypeptide but does not substantially recognize and bind to other non-NRAGE molecules in a sample, e.g., a biological sample, that naturally includes protein. A preferred antibody binds to an NRAGE polypeptide sequence of FIG. 1A or FIG. 1B.
By xe2x80x9cneutralizing antibodiesxe2x80x9d is meant antibodies that interfere with any of the biological activities of an NRAGE polypeptide, for example, the ability of NRAGE to bind to p75NTR. The neutralizing antibody may reduce the ability of an NRAGE to bind to p75NTR by 50%, more preferably by 70%, and most preferably by 90% or more. Any standard assay for the biological activity of NRAGE, including those described herein, may be used to assess potentially neutralizing antibodies that are specific for NRAGE.
By xe2x80x9cexposexe2x80x9d is meant to allow contact between an animal, cell, lysate or extract derived from a cell, or molecule derived from a cell, and a candidate compound.
By xe2x80x9ctreatxe2x80x9d is meant to submit or subject an animal (e.g. a human), cell, lysate or extract derived from a cell, or molecule derived from a cell to a candidate compound.
By xe2x80x9ccandidate compoundxe2x80x9d is meant a chemical, be it naturally-occurring or artificially-derived, that is assayed for its ability to modulate an alteration in reporter gene activity or protein levels, by employing one of the assay methods described herein. Test compounds may include, for example, peptides, polypeptides, synthesized organic molecules, naturally occurring organic molecules, nucleic acid molecules, and components thereof.
By xe2x80x9cassayingxe2x80x9d is meant analyzing the effect of a treatment, be it chemical or physical, administered to whole animals or cells derived therefrom. The material being analyzed may be an animal, a cell, a lysate or extract derived from a cell, or a molecule derived from a cell. The analysis may be, for example, for the purpose of detecting altered gene expression, altered RNA stability, altered protein stability, altered protein levels, or altered protein biological activity. The means for analyzing may include, for example, antibody labeling, immunoprecipitation, phosphorylation assays, and methods known to those skilled in the art for detecting nucleic acids.
By xe2x80x9cmodulatingxe2x80x9d is meant changing, either by decrease or increase, in biological activity.
By xe2x80x9ca decreasexe2x80x9d is meant a lowering in the level of biological activity, as measured by a lowering/increasing of: a) protein, as measured by ELISA; b) reporter gene activity, as measured by reporter gene assay, for example, lacZ/xcex2-galactosidase, green fluorescent protein, luciferase, etc.; c) mRNA, as measured by PCR relative to an internal control, for example, a xe2x80x9chousekeepingxe2x80x9d gene product such as xcex2-actin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH). In all cases, the lowering is preferably by 30%, more preferably by 40%, and even more preferably by 70%.
By xe2x80x9can increasexe2x80x9d is meant a rise in the level of biological activity, as measured by a lowering/increasing of: a) protein, measured by ELISA; b) reporter gene activity, as measured by reporter gene assay, for example, lacZ/xcex2-galactosidase, green fluorescent protein, luciferase, etc.; c) mRNA, as measured by PCR relative to an internal control, for example, a xe2x80x9chousekeepingxe2x80x9d gene product such as xcex2-actin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Preferably, the increase is by 5% or more, more preferably by 15% or more, even more preferably by 2-fold, and most preferably by at least 3-fold.
By xe2x80x9cpromoterxe2x80x9d is meant a minimal sequence sufficient to direct transcription. Also included in the invention are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell type-specific, tissue-specific, temporal-specific, or inducible by external signals or agents; such elements may be located in the 5xe2x80x2 or 3xe2x80x2 or intron sequence regions of the native gene.
By xe2x80x9coperably linkedxe2x80x9d is meant that a gene and one or more regulatory sequences are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequences.
By xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d is meant a carrier that is physiologically acceptable to the treated mammal while retaining the therapeutic properties of the compound with which it is administered. One exemplary pharmaceutically acceptable carrier is physiological saline solution. Other physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in Remington: The Science and Practice of Pharmacy, (19th edition), ed. A. Gennaro, 1995, Mack Publishing Company, Easton, Pa.